Non-M, non-O HIV-1 strains, fragments and uses

ABSTRACT

Retroviral strains of the non-M, non-O HIV-1 group, in particular a strain designated YBF30, its fragments and also its uses as a diagnostic reagent and as an immunogenic agent. The HIV-1 viruses which differ both from the M group and the O group exhibit the following characteristics: little or no serological reactivity with regard to the proteins of the M and O groups and strong serological reactivity with regard to the proteins which are derived from the strain YBF30 according to the invention or the strain CPZGAB SIV; absence of genomic amplification when using primers from the env and gag regions of the M and O HIV-1 groups; genomic amplification in the presence of primers which are derived from the YBF30 strain according to the invention; and homology of the products of the envelope gene which is greater than 70% with regard to the YBF30 strain.

This application is a divisional of application Ser. No. 10/301,661,filed Nov. 22, 2002, which is a divisional of application Ser. No.09/319,588, filed Aug. 27, 1999, which is the National Stage ofInternational Application No. PCT/FR97/02227, filed Dec. 8, 1997, whichclaims priority to France Application No. 96/15087 filed Dec. 9, 1996,which are herein incorprorated by reference in their entirety.

The present invention relates to retroviral strains of the non-M, non-OHIV-1 group, in particular a strain designated YBF30, to its fragmentsand to its uses as a diagnostic reagent and as an immunogenic agent.

The human acquired immunodeficiency viruses HIV-1 and HIV-2 areretrolentiviruses, which are viruses found in a large number of Africanprimates. All these viruses appear to have a common ancestor; however,it is very difficult to prejudge the period at which these differentviruses became separated from this precursor. Other viruses which aremore distant, but which nevertheless belong to the same group, are foundin other mammals (ungulates and felines).

All these viruses are associated with long infections; an absence ofsymptoms is the rule in monkeys which are infected naturally.

While the origin of HIV-2 appears to be clear on account of its stronghomology with the Sooty Mangabey (West Africa) virus, no virus which isclosely related to HIV-1 has been found in monkeys. The most closelyrelated viruses are viruses found in two chimpanzees (CPZGAB SIV, ANTSIV).

All the lentiviruses have been found to exhibit substantial geneticvariability, and the phylogenetic study of these variants, obtained froma large number of different geographic locations, has enabled 8 subtypes(clades) of HIV-1 to be distinguished, all of which are equidistant fromeach other. The clades are only a mathematical representation of theexpression of the variability: phenetic analysis, which is based on theamino acids rather than on the nucleic acids, gives different results(Korber et al., 1994).

The demonstration of subtypes is in accord with a phylogenetic analysiswhich does not, to date, have any pathophysiological correlation but,instead, a geographical correspondence. This is because each subtype ismainly found in a particular geographical area. The B subtype ispredominant in Europe and the United States whereas two subtypes, i.e. Eand B, are found in Thailand and there is a strong correlation betweenthe mode of transmission which, in actual fact, corresponds to aparticular population and the subtype found. All the clades have beenfound in Africa and their distribution across the rest of the worldreflects a probability of encounter between persons indulging inhigh-risk behaviour. The main clade, which is the main one because it ispresent in substantial proportions in Africa, is clade A. A very greatdegree of variability has been found in some African countries (G.Myers, 1994; P. M. Sharp et al., 1994). Several subtypes have beencharacterized in the western central African countries such as theCentral African Republic (Murphy et al., 1993) and Cameroon (Nkengasonget al., 1994).

Finally, patients have been characterized who are carriers of viralvariants of HIV-1, whose sera have posed detection problems forparticular kits which are sold on the French market and whoseconfirmatory Western blots have been atypical (Loussert-Ajaka et al.,1994; Simon et al., 1994; PCT International Application WO 96/27013).

Analysis of these variants has confirmed the fact that the type 1 HIVviruses should be subdivided into two groups, i.e. the M (major) groupand an O (outlier) group, which includes these isolates, as Charneau etal., 1994 had proposed. Analysis of the synonymousmutations/non-synonymous mutations ratio carried out on the sequences ofthe known O group viruses indicates that this new group is also ancient,even if no more ancient than the M group (Loussert-Ajaka et al., 1995).Its low prevalence to date, i.e. 8% of patients infected with HIV-1 inCameroon (Zekeng et al., 1994) and 18 cases characterized in France, isthought to be due to factors which are purely epidemiological.

These two groups of HIV-1 form a tree which is in the shape of a doublestar (FIGS. 9 to 19). Two isolates, i.e. CPZGAB SIV, characterized froma chimpanzee from Gabon (Huet et al., 1990) and CPZANT SIV,characterized from a chimpanzee in the Antwerp Zoo, possess sequencesand genetic organizations which are very closely related to HIV-1 butwhich do not fall within either of these two groups and form two newbranches on the phylogenetic tree.

The demonstration of new variants is important for developingsufficiently sensitive and specific reagents for detecting HIVinfections, that is to say reagents which do not lead to false-negativeor false-positive results, and for developing compositions which areprotective in regard to subtypes which do not belong either to the Mgroup or to the O group.

Consequently, the applicant has set itself the objective of providing anon-M, non-O strain, as well as sequences derived from this strain,which are suitable for detecting non-M and non-O HIV-1 variants andwhich do not lead to false-negative or false-positive results beingobtained. In order to do this, the inventors have, in particular,established an algorithm for differentiating between, and confirming,group M and group O HIV-1 infections, thereby enabling them to selectnon-M, non-O variants.

The present invention relates to a non-M, non-O HIV-1 strain whichexhibits the morphological and immunological characteristics of theretrovirus which was deposited on 2 Jul. 1996 under number I-1753(designated YBF30) in the Collection Nationale de Cultures deMicroorganismes (National Collection of Microorganism Cultures, 28 ruedu Docteur Roux, 75724 Paris Cedex 15), kept by the Pasteur Institute.

A non-M, non-O variant is understood as meaning a type 1 HIV whichcannot serologically and molecularly be recognized as belonging toeither of these groups.

The present invention also relates to the complete nucleotide sequenceof the strain as defined above (SEQ ID No. 1) as well as to nucleic acidfragments which are at least 10 nucleotides in size and which arederived from the said strain.

Fragments of this type which may be mentioned are: YBF 30 LTR (SEQ IDNo. 2), YBF 30 GAG (SEQ ID No. 3) (gag gene), YBF 30 POL (SEQ ID No. 5)(pol gene), YBF 30 VIF (SEQ ID No. 7) (vif gene), YBF 30 VPR (SEQ ID No.9) (vpr gene), YBF 30 VPU (SEQ ID No. 11) (vpu gene), YBF 30 TAT (SEQ IDNo. 13) (tat gene), YBF 30 REV (SEQ ID No. 15) (rev gene), YBF 30 ENVgp160 (SEQ ID No. 17) (env gene), YBF 30 NEF (SEQ ID No. 19) (nef gene),

-   -   the SEQ ID Nos. 21-57, also designated, respectively, YLG,        LPBS.1, GAG Y AS1.1, GAG Y AS1, GAG 6, GAG Y S1, GAG Y S1.1, GAG        Y S1.2, YRT AS1.3, YRT AS1.2, YRT AS1.1, YRT 2, YRT AS1, YRT        2.1, YRT 2.2, YRT 2.3, YRT 2.4, 4481-1, 4481-2, 4235.1, 4235.2,        4235.3, 4235.4, SK69.6, SK69.5, SK69.4, SK69.3, SK69.2, SK69.1,        SK68.1, SK68.2, SK68.3, LSI AS1.3, LSI AS1.2, LSI AS1.1, LSI Al,        YLPA, as well as any sequence which is not identical to one of        the above nucleotide sequences or is not complementary to one of        these sequences but is nevertheless capable of hybridizing        specifically with a nucleic acid sequence derived from a non-M,        non-O HIV-1 virus.

Such sequences can be used in the specific identification of a non-M,non-O HIV-1, and as diagnostic reagents, either alone or pooled withother reagents, for the differential identification of any HIV-1.

These sequences may, in particular, be employed in diagnostic testswhich comprise either a direct hybridization with the viral sequence tobe detected or an amplification of the said viral sequence, with thesetests using, as primers or as probes, an oligonucleotide which comprisesat least 10 nucleotides and which is included in any one of the abovesequences, in particular one of the abovementioned sequences, SEQ IDNos. 21-57.

The present invention also relates to HIV-1 viruses which arecharacterized in that they differ both from the M group and from the Ogroup and exhibit the following characteristics:

-   -   little or no serological reactivity with regard to proteins of        the M and O groups and strong serological reactivity with regard        to proteins which are derived from the YBF30 strain or the        CPZGAB SIV strain;    -   absence of genomic amplification when using primers from the env        and gag regions of HIV-1 viruses of the M and O groups;    -   genomic amplification in the presence of primers which are        derived from the YBF30 strain, as defined above; and    -   homology of the products of the envelope gene which is >70% with        regard to the YBF30 strain.

The invention also relates to the use of the above described sequencesfor implementing a method of hybridization and/or of gene amplificationof nucleic acid sequences of the HIV-1 type, with these methods beingapplicable to the in-vitro diagnosis of the potential infection of anindividual with a virus of the non-M, non-O HIV-1 type.

This in-vitro diagnostic method is carried out using a biological sample(serum or circulating lymphocyte) and comprises:

-   -   a step of extracting the nucleic acid which is to be detected        and which belongs to the genome of the virus, which virus may        possibly be present in the biological sample, and, where        appropriate, a step of treating the nucleic acid using a reverse        transcriptase, if this nucleic acid is in RNA form,    -   at least one cycle comprising the steps of denaturing the        nucleic acid, of hybridizing with at least one sequence in        accordance with the invention and, where appropriate, extending        the hybrid, which has been formed, in the presence of suitable        reagents (polymerizing agent, such as DNA polymerase and dNTP),        and    -   a step of detecting the possible presence of the nucleic acid        belonging to the genome of a virus of the non-M, non-O HIV-1        group type.

The following conditions are employed for the PCR using the primersderived from the YBF30 strain:

-   -   extracting the lymphocytic DNA by means of the phenol/chloroform        technique and quantifying it by spectrophotometry at a        wavelength of 260 nm. All the amplifications are carried out        using a Perkin Elmer 2400 thermocycler.    -   the long (9 kb) PCRs are carried out using an XL PCR kit (Perkin        Elmer) in accordance with the manufacturer's conditions and        using the dNTP's, the buffers provided and Perkin Elmer's “hot        start”; the amplification cycles of this long PCR are:    -   1 cycle of denaturation for 2 minutes at 94° C.,    -   then 16 cycles: 15 seconds at 94° C., 15 seconds at 55° C., 8        minutes at 68° C.,    -   then 24 cycles: 15 seconds at 94° C., 15 seconds at 55° C., 8        minutes at 68° C., adding a further 15 seconds (incrementation)        to each cycle.    -   the nested PCRs are carried out on the amplification products of        the long PCRs. The conditions for carrying out the nested PCRs        are as follows:    -   “Expand High Fidelity PCR System” Taq polymerase buffer and        enzyme from Boehringer Mannheim in accordance with the        manufacturer's instructions, dNTP and “hot start” from Perkin        Elmer,    -   200 μmol of each dNTP, 20 pmol of each primer in accordance with        the invention, 5 μl of DNA, 10 μl of 10 ×PCR buffer and 2.6        units of Taq polymerase in a volume of 100 μl,    -   amplification: one cycle of 2 minutes at 940° C. followed by 38        cycles: 15 seconds at 940° C., 15 seconds at 550° C., a time of        elongation at 72° C. which varies in accordance with the size of        the PCR product to be amplified (from 30 seconds to 2 minutes)        and a final elongation cycle of 10 minutes at 72° C.

The amplified product is preferably detected by direct sequencing.

The invention also relates to a peptide or a peptide fragment which ischaracterized in that it can be expressed by a non-M, non-O HIV-1 strainor using a nucleotide sequence as defined above, and in that it iscapable: (1) of being recognized by antibodies which are induced by anon-M, non-O HIV-1 virus, as defined above, in particular the YBF30strain or a variant of this strain, and which are present in abiological sample which is obtained following an infection with a non-M,non-O HIV-1 strain, and/or (2) of inducing the production of anti-non-M,non-O HIV-1 antibodies.

Peptides of this type which may be mentioned are, in particular, thosewhich are derived from the YBF30 strain, in particular: that which isexpressed by the gag gene (SEQ ID No. 4), that which is expressed by thepol gene (SEQ ID No. 6), that which is expressed by the vif gene (SEQ IDNo. 8), that which is expressed by the vpr gene (SEQ ID No. 10), thatwhich is expressed by the vpu gene (SEQ ID No. 12), that which isexpressed by the tat gene (SEQ ID No. 14), that which is expressed bythe rev gene (SEQ ID No. 16), that which is expressed by the env gene(SEQ ID No. 18), or one of its fragments such as a fragment of the V3loop region, i.e. CTRPGNNTGGQVQIGPAMTFYNIEKIVGDIRQAYC (SEQ ID No. 58),and that which is expressed by the nef gene (SEQ ID No. 20), or afragment of these peptides which are capable of recognizing theantibodies which are produced during an infection with a non-M, non-OHIV-1 as defined above.

The invention also relates to immunogenic compositions which compriseone or more translation products of the nucleotide sequences accordingto the invention and/or one of the peptides as defined above, obtained,in particular, by synthetic means.

The invention also relates to the antibodies which are directed againstone or more of the above-described peptides and to their use forimplementing methods for the in-vitro, in particular differential,diagnosis of the infection of an individual with a virus of the HIV-1type using methods which are known to the skilled person.

The present invention encompasses all the peptides which are capable ofbeing recognized by antibodies which are isolated from an infectiousserum which is obtained after an infection with a non-M, non-O HIV-1strain, and the peptides which are capable of being recognized by anantibody according to the invention.

The invention furthermore relates to a method for the in-vitro diagnosisof a non-M, non-O HIV-1 virus, which method is characterized in that itcomprises bringing a biological sample, which has been taken from apatient, into contact with antibodies according to Claim 10, which maypossibly be combined with anti-CPZGAB SIV antibodies, and detecting theimmunological complexes which are formed between the HIV-1 antigens,which may possibly be present in the biological sample, and the saidantibodies.

The invention also relates to a kit for diagnosing HIV-1, which kit ischaracterized in that it includes at least one reagent according to theinvention.

Apart from the provisions which have been described above, the inventionalso comprises other provisions which will be evident from thedescription which follows and which refers to examples of implementingthe method which is the subject of the present invention and also to theattached drawings, in which:

FIGS. 1 to 7 illustrate the location of the different primers on thegenome of the YBF30 strain;

FIG. 8 illustrates the genomic organization of the YBF30 strain;

FIGS. 9 to 16 depict the phylogenetic analysis of the different genes ofthe YBF30 strain as compared with group M HIV-1 and group O HIV-1 (FIG.9: ltr gene, FIG. 10: gag gene, FIG. 11: tat gene, FIG. 12: rev gene,FIG. 13: vif gene, FIG. 14: env gp120 gene, FIG. 15: env gp41 gene, FIG.16: nef gene, FIG. 17: pol gene, FIG. 18: vpr gene, FIG. 19: vpu gene);

FIG. 20 illustrates the percentage genetic distance between YBF30 andHIV-1/CPZGAB SIV.

It should of course be understood, however, that these examples aregiven solely by way of illustrating the subject-matter of the invention,of which they in no way constitute a limitation.

EXAMPLE Obtaining a Non-M, Non-O HIV-1 Variant According to theInvention (YBF30) and Its Uses

This was, in particular, possible in connection with studying theepidemiology of infection with human acquired immunodeficiency viruses(HIV) in Cameroon, which epidemiology is especially paradoxical. In thiscountry, the diversity of the strains is remarkable as most of thesubtypes of the M (major) group of HIV-1 viruses known to date have beenreported. Cases of infection with highly divergent HIV-1 viruses of theO group (O for outlier) have been reported, almost exclusively inpatients of Cameroonian origin. Cases of infection with HIV-2, HTLV-1and HTLV-2 subtypes A and B have also been reported.

Taking as a basis the results of previous serological and genotypicassessments, the inventors established an algorithm for differentiatingbetween and confirming infections with HIV-1 viruses of the M and Ogroups in order to select non-M, non-O variants.

These methods were applied to samples which were sent to the NationalReference Laboratory for HIV infections at Yaoundé and made it possibleto characterize a highly divergent HIV isolate and to define the toolsfor characterizing a new HIV-1 group, taking into account the homologieswhich were observed between this human strain YBF30 and the simianstrain CPZGAB SIV.

I—Way of Serologically Characterizing the YBF30 Variant During theEpidemiological Study

1) Collecting the Samples:

All the adult patient sera which were sent to the Yaoundé referencelaboratory in 1994 and 1995 for detecting or confirming an HIV infectionwere studied (n=8831).

2) Differentiating Serologically Between Group M and Group HIV-1, andSelecting Variants:

If there was positive detection of anti-HIV antibodies (Génélavia Mixtindirect mixed HIV-1 and HIV-2 EIA, Sanofi-Pasteur, Paris, France), thiswas then combined with an EIA test based on the principle of competitionwith a specific antigen of the M group (Wellcozyme Rec HIV-1, Murex,Dartford, UK).

If the competitive Wellcozyme Rec HIV-1 test is positive, with a ratiofor the reactivity in optical density (OD) as compared with thethreshold or cut-off (CO) value which is greater than 5 (CO/OD>5), theserum is regarded as being HIV-1-positive, a result which should beconfirmed on a new sample.

The choice of a reactivity ratio which is greater than 5 for regardingthe competitive test as being a test for confirming infection with HIV-1is based on experience acquired by the virology laboratory of Bichathospital: all of 7200 samples which reacted with a ratio >5 gave astrongly positive HIV-1 Western blot (WB, New Lav Blot 1, SDP, Marnes laCoquette). Apart from cases of HIV-1 seroconversion, the samples whichare confirmed as being HIV-positive and which give a Wellcozyme ratio of<5 correspond either to infections with HIV-2 or to infections with Ogroup HIV-1 or other HIV-1 variants.

In order to eliminate the false-positive reactions when carrying out amixed EIA detection, the samples which give a CO/OD ratio of <5 aretested systematically with a third generation mixed HIV-1/HIV-2 EIA(Enzygnost Plus, Marburg, Germany) which includes antigens of the M andO HIV-1 groups (recombinant gp41 of the MVP5180 strain). If this test ispositive, a rapid test which discriminates between HIV-1 and HIV-2(Multispot, SDP, Marnes la Coquette) and a Western blot (WB, New LavBlot 1 or 2, SDP) are then carried out.

3) Serologically Confirming Infections with O Group HIV-1 and HIV-1Variants.

All the samples which give a CO/OD ratio of <5, and which have beendifferentiated as being positive by WB (positivity criteria: 2ENV+/−POL+/−GAG or 1 ENV+POL+/−GAG) and HIV-1, are tested with a dotblot test using peptide antigens of the V3 and transmembrane regions(InnoLia, Innogenetics, Ghent, Belgium).

4) Retroviral Isolation of the Group O Variant Strains.

The peripheral blood mononuclear cells (PBMC) from the seropositivepatients were isolated by Ficoll-Hypaque gradient in Cameroon and thenstored, and transported to Paris, in liquid nitrogen.

After thawing, the PBMCs from the patients were cocultured together withlymphocytes from seronegative Caucasian donors. Viral replication in theculture supernatants was demonstrated by detecting reverse transcriptaseactivity and by carrying out tests for detecting the p24 antigen (Elaviap24 polyclonal, SDP) over a period of one month.

5) Sequences:

The PCR products are visualized on agarose gels of from 1 to 1.4%concentration, depending on the size of the fragments, precipitated in3M sodium acetate (1:10) and 3 volumes of absolute ethanol, incubated at−80° C. for 30 minutes and then centrifuged at 13,000 rpm for 20minutes. The pellet is dried and then taken up in 10 μl of distilledwater (Sigma). Purification is carried out on a “Qiaquick Gel Extractionkit” (Qiagen) in accordance with the manufacturer's instructions; theproducts are sequenced on an automated DNA sequencer (AppliedBiosystems, Inc., Foster City, Calif.) using an Applied Biosystem DyeTerminator kit, as previously described (Loussert-Ajaka et al., 1995);the nucleotide sequences are analysed on Sequence Navigator software(Applied Biosystems), and aligned using GeneWorks software(Intelligenetics Inc.).

6) Phylogenetic Analyses:

The sequences were aligned using the CLUSTAL software for multiplealignments and taking, as the reference matrix, the alignments of thecompilation of HIV sequences possessed by the Laboratory of Biology andTheoretical Biophysics, Los Alamos, N. Mex., 87545 USA.

The phylogenetic analyses were performed using the PHYLIP software; thedistances were firstly calculated using DNADIST, after which thephylogenetic analysis was carried out using NEIGBOR JOINING or FITCH;finally, the trees were drawn using DRAWTREE (FIGS. 9 to 19). Thegenetic distance percentages are also shown in FIG. 20.

SEQBOOT was first of all used for the “bootstrapping” analyses, followedby DNADIST and NEIGHBOR JOINING or FITCH. Finally, the bootstrap valueswere obtained using CONSENS.

II—Results of the Investigation for Detecting Group O and Variant HIVViruses:

174 samples, out of 3193 samples found to be positive in the screening,were regarded as being group O or group M with abnormal serologicalreactivity or as being variants.

III—Detection of a Non-group O and Non-group M Sample ExhibitingAbnormal Serological Reactivity

The 174 sera which were HIV-1-positive by WB (Western blot), butreactive with a CO/OD ratio of <5 in the competitive EIA, were tested bydifferential LIA dot blot on the V3 peptides from group M, group O andCPZGAB SIV:

-   -   7 do not react with any of the peptides represented (M, O or        CPZGAB SIV). The absence of any cell collection does not allow        any conclusion to be drawn.    -   82 give a reactivity with regard to at least one of the peptides        corresponding to the V3 loop of O group strains. The frequency        of the crossreactions is low and restricted to the epitopes        which correspond to the consensus V3 regions (11%) and to the        CPZGAB SIV V3 regions (43%).    -   84 sera do not react with the O group epitopes. Most of these        samples were obtained from patients exhibiting an AIDS syndrome        (75/84).    -   one serum, which was taken from a Cameroonian patient (NJ)        reacts exclusively with the CPZGAB SIV peptide. This isolated        reactivity with regard to a CPZGAB SIV antigen has never been        described previously. Since lymphocytes had been collected from        the patient, it was possible to continue with the virological        characterization of this strain, which was termed YBF30.        IV—Results of the Serological and Virological Examinations        Performed on the First Samples Taken from this Patient        (May 1995) (Serum No.: 95-6295):

1) Commercial ELISA Tests (Optical Density/Threshold Value)

Criterion of positivity: OD/CO>1

Génélavia=>15

Wellcozyme CO/OD=1.55

Abbott Plus=>15

Behring Plus=4.2

2) Western Blot

New Lav 1 Pasteur WB:

160++, 120++, 68++, 55+, 41+, 40+/−, 34++, 24++, 18+

3) Innogenetics LIA Dot Blot

Negative for all the group O and group M bands apart from CPZGAB SIV V3

4) Results of the Investigative Serological Examinations Carried Put onPeptides Which Are Specific for the M and O Groups

The technique developed by Professor Francis Barin of the VirologyLaboratory of the Tours CHU was modified (Barin F. et al., 1996); usewas made of synthesized transmembrane region peptides (BioMérieux) fordeveloping a test for differentiating between the M and O groups. Thistechnique is based on antibody-binding competition between thetransmembrane gp41 peptides of the O and M groups, which are depositedon the solid phase, and gp41 transmembrane peptides either of the Ogroup or of the M group at higher concentration in a hyperosmolar liquidreaction phase. The results are shown in Table I below, in which the CPwell corresponds to the 100% inhibition control and the CSP wellcorresponds to the 0% inhibition control. TABLE I Results of theinter-group O-group M differentiations for the 6295 serum gp41 M gp41 OCP CSP 6295 0.25 0.36 0.12 1.98

These results demonstrate that there is strong binding with regard tothe peptides of the solid phase (CSP) and a marked inhibition due to thecombined addition of the M and O peptides (CP), but no cleardifferentiation either by the M peptide or by the O peptide. This is,therefore, serological evidence that the infecting strain does notbelong either to the M group or to the O group.

-   -   In view of an isolated reactivity in the InnoLia dot blot with        regard to the CPZGAB SIV V3 antigens, on the same bases of        competition between peptides, this serum was studied by bringing        into competition the gp41 M, gp41 O and gp41 CPZGAB SIV        peptides.

Use of the serum from the chimpanzee named ‘Amandine’ (donated by M.Peeters, who isolated the CPZGAB SIV strain, AIDS 1992) initiallyenabled this technique to be validated. In Table II, the lowest values(OD) indicate the highest degree of binding to the antigens. TABLE IIResults of the inter-group O-group M-CPZGAB SIV differentiations usingthe Amandine chimpanzee serum and the 6295 serum gp41 gp41 M gp41 OCPZGAB CP CSP Amandine 0.8 1.4 0.3 0.5 1.9 6295 0.7 1.1 0.7 0.4 2.1

The reactivity of the “Amandine” serum confirms and validates the testaccording to the invention and shows that, while the serum of thepatient reacts identically with regard to the M and CPZGAB SIV peptides,it does not exhibit a crossreaction with the O peptide.

These results demonstrate that the group M gp41 and CPZGAB SIV gp41peptides exert a similar inhibition on the serum of the patient. Theantigens of the infecting strain have therefore given rise to antibodieswhich recognize the group M and CPZGAB SIV gp41 peptides in a similarmanner.

4) Results Obtained from the Lymphocyte Isolation (Sampling of May 1995)

A retrovirus was isolated, using standard techniques, from thelymphocytes which were sampled on 22 May 1995. Culture using the MT2cell line shows that the YBF30 strain does not form any syncytia (NSI).

V—Results of the Serological Examinations Carried Out on the SecondBlood Sample (November 1995) (Serum No. 95-3371)

1) Innogenetics LIA Dot Blot

Negative for all the bands, apart from CPZGAB SIV V3

2) Results of the Investigative Serological Examinations Carried Out onthe Peptides Specific for the M and O Groups.

Table III shows the results of the inter-group O-group M-CPZGAB SIV gp41differentiations using the 3371 serum. TABLE III Results of theinter-group O-group M-CPZGAB SIV gp41 differentiations using the 3371serum gp41 M gp41 O gp41 CPZGAB CP CSP 3371 1.31 1.7 0.89 0.54 2.02

These results confirm, on this new blood sample (taken from the samepatient in the terminal stage of the disease), that the CPZGAB SIV gp41peptide markedly inhibits the serum of the patient.

The antigens of the infecting strain have therefore induced antibodieswhich preferentially recognize the CPZGAB SIV gp41 peptide.

3) Results from the Lymphocyte Isolation (Blood Sampling of November 95(95-3371-YBF31))

A retrovirus was isolated, using the standard techniques, from thelymphocytes which were sampled in November 1995 and termed YBF31; thesequence elements are identical to those of YBF30.

VI—Genomic Amplification and Sequences of YBF30

The DNA for all the PCR manipulations is extracted from the cellsobtained at the end of a positive culture.

The PCRs carried out using the O group HIV-1 primers are negative in thedifferent regions tested (gag, pol, env). Similarly, those carried outusing the primers which are specific for M group HIV-1 are alsonegative.

The amplification and hybridization conditions for the O group PCRs arethose described in Loussert-Ajaka, 1995. The amplification andhybridization conditions for the M group PCRs are those described by theauthors cited below.

These M group primers are located in accordance with the HIV-1 HXB2sequence as follows:

-   -   in env gp120: ED3/ED12 (position 5956-5985; 7822-7792); ED5/ED14        (6556-6581; 7960-7931); ED5/ED12; ED3/ED14; ES7/ES8 (7001-7020;        7667-7647) (Delwart et al. Science 1993; 262: 1257-1261).

in env gp41 first PCR, ED3/M29, followed by a nested PCR, M28/M29(7785-7808; 8099-8124); M28/M29 have the following sequences: M28:CGGTTCTT(AG)GGAGCAGC(ACT)GGAAGCA, (SEQ ID NO: 99) M29:T(CT)T(ACGT)TCCCA(CT)T(AT)(CT)A(AGT)CCA(AGT) GTCAT; (SEQ ID NO: 100)

SK68/SK69 (Ou et al. Science, 1988; 239: 295-297).

-   -   in gag: Amplicor Roche Diagnostics systems; nested gag primers        (Loussert-Ajaka et al. Lancet 1995; 346: 912-913); SK38/SK39 (Ou        et al., Science, 1988; 239: 295-297).    -   in pol: A/NE1 (Boucher et al., Lancet, 1990; 336: 585-590);        Pol3/Pol4 (Laure et al., Lancet, 1988, ii, 538-541).

Only the PCRs carried out using the H Pol primers (4235/4538) arepositive, with this being followed by a nested PCR using the primers4327/4481 (Fransen et al., Molecular and Cellular Probes 1994; 8:317-322). This H Pol fragment, which is located in the integrase (260bp), has been sequenced. Amplification using the HPOL primers is madepossible due to the excess of virus. This is because the DNA which isused is extracted from cells at the end of a strongly positive culture(reverse transcriptase>100,000 cpm). It is not possible to amplify theDNA which is extracted from fresh cells without coculture because of thelarge number of mispairings between the HPOL primers (especially in the3′ region) and the sequence of the YBF30 isolate. Conservation of this3′ end is very important for the extension activity of the Taqpolymerase.

1—Sequence of the pol gene: the use of very degenerate primers foramplifying, by RT-PCR, the RNA extracted from the positive culturesupernatant gave a positive amplification. These are primers which arecommon to all retroviruses (Donehower et al. J. Virol. Methods 1990; 28:33-46), and are located in the reverse transcriptase region of the polgene. Analysis of the fragment after sequencing made it possible togenerate a specific primer, i.e. YRT2 (SEQ ID No.32), from the YBF30isolate and to amplify the pol gene using the Hpol 4481 primer (Fransenet al., 1994, loc. cit.) as the antisense primer. The fragment wassequenced by synthesizing specific primers as required for each fragmentgenerated (FIG. 1).

2—Sequence of the env gene: the second approach was to perform a longPCR (XL-PCR, Perkin Elmer), thereby amplifying all the virus (9000 bp)using primers situated in the LTR: LPBS 1 (SEQ ID No.22); LSiGi,followed by a 6000 bp nested PCR using YRT2 (SEQ ID No.32)/SK69, and tosequence all the envelope following the same procedure. The gp41 regionwas sequenced using a nested PCR and employing the primers SK68/LSiGi.

3—Sequence of the gag gene: use of a nested PCR, achieved by means of along PCR (LPBS 1/LSiGi), employing the primers Gag 5 and Gag 11i, andgenerating from this specific primers, as required, in order to walkalong the viral genome.

VII—Results of the Sequencings

The strain YBF30 was sequenced completely (see list of sequences). TheYBF31 strain of November 1995 was sequenced in part, and the absence ofsignificant variation confirms the validity of the YBF30 sequences.

VIII—Synthesizing Peptides of the V3 Loop Region of the YBF30 Strain.

Studying the sequences of the V3 loop region made it possible tosynthesize the corresponding peptide and to compare the amino acids ofthis region of the YBF30 strain with those of other M subtypes and Ostrains.

The sequences of the peptides are: YBF30: SEQ ID No. 58 CPZGAB SIV:CHRPGNNTRGEVQIGPGMTFYNIENVYGDTRSAYC (SEQ ID No. 59) GROUP O:CIRPGNRTYRNLQIGPGMTFYNVEIATGDIRKAFC (ANT70) (SEQ ID No. 60) GROUP M:CTRPNNNTRKSVRIGPGQAFYATGDIIGDIRQAHC (SS-TYPE A) (SEQ ID No. 61)

The peptide was synthesized, starting with the two asparagines of the 5′region of the loop, and used in accordance with the same principle aspreviously described (see IV 4)), namely in competition in relation tothe peptides of the M group, the O group and CPZGAB SIV. The resultsshown in Table IV confirm the original nature of this strain and thepossible spread of these strains, since the serological results favourinfection of the YBF30 type in Cameroon. Furthermore, a study of 200selected HIV-1-positive sera from Cameroon provides evidence of a newcase exhibiting a profile which is similar to that of YBF30. TABLE IVStudy of the reactivity of 200 sera Serum Origin V3A V3cpz V3YBF30 CPCSP 953371 Cameroon 1.66 0.38 1.39 0.39 1.64 956295 Cameroon 1.72 0.371.16 0.51 1.73 967321 Cameroon 0.07 0.17 0.5 0.05 0.27 Amandine GABSIV1.74 0.14 1.48 0.19 1.74 NOA.* ANTSIV 2.66 0.31 1.88 0.46 1.9*serum from CPZ ANT SIV

The reactivity of the sera 953371 and 956295, corresponding to thepatient from whom the YBF30 strain was isolated, with the CPZ SIVpeptide, was confirmed in this new test. The lower reactivity withregard to its own V3 antigen is usual during the late stages of thedisease. Nevertheless, this reactivity remains greater than that raisedwith regard to the M peptide. Another Cameroonian patient (serum 967321)exhibits the same profile of peptide reactivity.

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Schneider-Fauveau, J. de Saint-Martin, I.

Loussert-Ajaka, M. L. Chaix, S. Saragosti, A. M.

Couroucé, D. Ingrand, C. Janot, and F. Brun-Vezinet. AIDS, 1994, 8,1628-1629. Sensitivity of screening kits for anti-HIV-1 subtype Oantibodies.

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As is evident from the above, the invention is in no way limited tothose of its embodiments which have just been described more explicitly;on the contrary, it encompasses all the variants which may come to themind of the skilled person without departing from the context or scopeof the present invention.

1-14. (canceled)
 15. A non-M, non-O HIV-1 strain which exhibits themorphological and immunological characteristics of the retrovirusdesignated YBF30 deposited as CNCM number I-1753.
 16. A nucleic acidsequence derived from the strain according to claim
 15. 17. A nucleicacid sequence according to claim 16, characterized in that it isselected from the group consisting of the following sequences: thecomplete nucleotide sequence of the strain (SEQ ID NO: 1) as well asnucleic acid fragments which are derived from the said strain: (SEQ IDNO: 2), (SEQ ID NO: 5), (SEQ ID NO: 7), (SEQ ID NO: 9), (SEQ ID NO: 11),(SEQ ID NO: 13), (SEQ ID NO: 15), (SEQ ID NO: 17), (SEQ ID NO: 19), andthe sequences SEQ ID NOs: 21, 22, and 29-57, and any sequence which isnot identical to one of the above nucleotide sequences, or is notcomplementary to one of these sequences, but is nevertheless capable ofhybridizing with a nucleic acid sequence which is derived from a non-M,non-O HIV-1 virus.
 18. An oligonucleotide, characterized in that it isselected from the sequences SEQ NOs: 21 to 57, and in that it is capableof being used as a primer or as a probe for detecting an HIV-1 accordingto claim
 15. 19. An HIV-1 virus, characterized in that it differs bothfrom the M group and from the O group and exhibits the followingcharacteristics: little or no serological reactivity with regard toproteins of the M and O groups and strong serological reactivity withregard to proteins which are derived from the YBF30 strain according toclaim 15 or the CPZGAB SIV strain; absence of genomic amplification whenusing primers from the env and gag regions of the HIV-1 viruses of the Mand O groups; genomic amplification in the presence of the primers whichare derived from the YBF30 strain, and selected from the groupconsisting of SEQ ID NOs: 21 to 57; homology of the products of theenvelope gene which is greater than 70% with regard to the YBF30 strain.20. A method for diagnosing in vitro an HIV-1 virus of the non-M, non-Ogroup by means of hybridization and/or gene amplification, which methodis carried out using a biological sample and is characterized in that itcomprises: a step of extracting viral nucleic acid which is to bedetected from a biological sample and, where appropriate, a step oftreating the nucleic acid using a reverse transcriptase, if this nucleicacid is in RNA form; at least one cycle comprising the steps ofdenaturing the nucleic acid, followed by hybridizing with at least onesequence according to claim 17 and, where appropriate, extending thehybrid, which has been formed, in the presence of suitable reagents, anda step of detecting any nucleic acid belonging to the genome of a virusof the non-M, non-O HIV-1 group type.
 21. Antibodies which are directedagainst one or more of the peptides that can be expressed by a non-M,non-O HIV-1 strain according to claim 15 wherein the one or morepeptides are capable of being recognized by antibodies which are inducedby a non-M, non-O HIV-1 virus and which are present in a biologicalsample which is obtained following an infection with a non-M, non-OHIV-1 strain.
 22. Antibodies which are directed against one or more ofthe peptides that is selected from the group consisting of that which isexpressed by the gag gene (SEQ ID NO: 4), that which is expressed by thepol gene (SEQ ID NO: 6), that which is expressed by the vif gene (SEQ IDNO: 8), that which is expressed by the vpr gene (SEQ ID NO: 10), thatwhich is expressed by the vpu ene (SEQ ID NO: 12), that which isexpressed by the tat gene (SEQ ID NO: 14), that which is expressed bythe rev gene (SEQ ID NO: 16), and that which is expressed by the nefgene (SEQ ID NO: 20), and fragments of these peptides which exhibit saidantibody-recognition capacity.
 23. A method for the in-vitro diagnosisof a non-M, non-O HIV-1 virus, characterized in that it comprisesbringing into contact a biological sample with antibodies according toclaim 22, and detecting the immunological complexes which are formedbetween any HIV-1 antigens, present in the biological sample and thesaid antibodies.
 24. A method as set forth in claim 23 wherein saidantibodies further comprise anti-CPZGAB SIV antibodies.
 25. A reagentfor diagnosing a non-M, non-O HIV-1 virus, characterized in that itcomprises a sequence according to claim
 17. 26. A method for screeningand typing a non-M, non-O HIV-1 virus, characterized in that itcomprises bringing any one of the nucleotide fragments according toclaim 17 into contact with the nucleic acid of the virus to be typed anddetecting any hybrid which is formed.
 27. A kit for diagnosing a non-M,non-O HIV-1 virus, characterized in that it includes at least onereagent according to claim 25.